The knowledge that elements in the soil influence root nodulation has long been recognized. Indeed, the Romans transferred soil from successful legume fields to unsuccessful ones in order to improve the quality of the latter.
It has since then been demonstrated that one important soil element responsible for nodulation is soil bacteria. The family Rhizobiaceae consists of a heterogeneous group of gram-negative, aerobic, non-spore-forming rods that can invade and induce a highly differentiated structure, the nodule (on the roots, and in some instances, stems of leguminous plants), within which atmospheric nitrogen is reduced to ammonia by the bacteria. The family Rhizobiaceae contains three genera, Rhizobium, Bradythizobium, and Azorhizobium. The host plant is most often of the family Leguminosae. The slow-growing nodulation bacteria which have specific associations with soybean are referred to as Bradyrizobium. Currently, Bradyrhizobium has only one named species B. japonicum, with others lumped together in a miscellaneous group (Barbour et al., 1992); these latter strains are reformed to as B. sp., followed by the plant species they infect in parenthesis. Some soybean plants can also nodulate with the fast growing Rhizobium fredii (Sprent and Sprent, 1990). Rhizobium species, sometimes designated "fast-growing" rhizobia, include among others R. meliloti which infects alfalfa.
The element N is essential to all living organisms because it is a component of many biologically important molecules. The most important of these include nucleic acids, amino acids and therefore proteins, and porphyrins, which occur in large amounts in all living cells. To be able to multiply and grow, or just survive, organisms require a source of N. The ability to reduce atmospheric dinitrogen is limited to prokaryotes. Legumes and a few other plant species have the ability to fix atmospheric N through symbiotic relationships. In the case of legumes N.sub.2 -fixation is carried out by prokaryotes, Rhizobium or Bradyrhizobium in nodules located on the plant root (Sprent and Sprent, 1990).
Nodulation and the development of effective symbiosis is a complex process requiring both bacterial and plant genes. The molecular mechanisms of recognition between (Brady)rhizobium and legumes can be considered as a form of cell-to-cell interorganismal communication. A precise exchange of molecular signals between the host plant and rhizobia over space and time is essential to the development of effective root nodules. The first apparent exchange of signals involves the secretion of phenolic compounds, flavonoids and isoflavonoids, by host plants (Peters and Verma, 1990). These signal compounds are often excreted by the portion of the root with emerging root hairs, a region that is most susceptible to infection by rhizobia (Verma, 1992). These compounds have been shown to activate the expression of nod genes in rhizobia, stimulating production of the bacterial nod factor (Kondorosi, 1992). This nod factor has been identified as a lipo-oligosaccharide (Carlson et al., 1993), able to induce many of the early events in nodule development, including deformation and curling of plant root hairs, the initiation of cortical cell division, and induction of root nodule meristems. In soybean for example, the isoflavones, daidzein and genistein, are the major components of soybean root exudates which induce the nod genes of B. japonicum (Kosstak et al., 1987). Other such substances active at very low concentrations (10.sup.-6 to 10.sup.-7 M) have been shown to stimulate bacterial nod gene expression within minutes. However, the effectiveness of isoflavonoids is found to vary between cultivars.
The "common" nod genes, designated nodA, B and C, which are associated with the early stages of infection and nodulation, are structurally conserved among Rhizobium strains. In R. meliloti, R. leguminosarum, and R. trifolii, the nodA, B and C genes are organized in a similar manner and are believed to be coordinately transcribed as a single genetic operon. The DNA region adjacent and 5' to nodA has been found to contain a fourth nodulation gene, designated nodD, which is transcribed divergently from the nodABC operon. NodD has been found to function in the regulation of expression of nodABC and other nodulation genes.
Comparisons of the DNA sequences and the deduced amino acid sequences of the encoded nodD product confirm the presence of significant sequence conservation of these genes among strains of Rhizobium. nodD mutants in the various species of Rhizobium do not, however, display the same nodulation phenotypes. It now appears that many species of Rhizobium carry multiple nodD-like genes, on their Sym plasmids.
Another similarity in the nod region(s) of Rhizobium strains is the presence of conserved sequence elements within the promoter regions of certain inducible nod genes. These conserved sequences, first identified in the nodABC promoter region, are termed the nod-box and are believed to function in induced nod gene expression, possibly as regulatory protein binding sites.
No Sym plasmids have been associated with Bradyrhizobium strains. The nitrogenase and nodulation genes of these bacteria are encoded on the chromosome. Of importance, Bradyrhizobium strains contain nodulation genes which are reported to functionally complement mutations in Rhizobium and which show significant structural homology to nodulation gene regions of R. meliloti and R. leguminosarum.
In Bradyrhizobium japonicum strains USDA 110 and 123, nodABC and D gene structural homologs have been identified which are organized in a manner similar to their homologs in Rhizobium strains. NodD is read divergently from nodAB and C which are organized in a single operon. The untranscribed region between nodD and nodA also contains a copy of the conserved nod-box.
In contrast to Rhizobium strains, Bradyrhizobium japonicum strains contain another open reading frame, designated ORF, between nodD and nodA which is believed to be part of the nodABC operon.
The specific components of legume exudate that act to induce nodulation genes in several species of Rhizobium and Bradyrhizobium have been identified as flavonoids. Luteolin was reported to be the component of alfalfa exudates that induces nodABC expression in R. meliloti. Three clover exudate constituents: 4',7-dihydroxyflavone, geraldone and 4'-hydroxy-7-methoxyflavone were reported to induce the nodulation genes of R. trifolii. Two pea exudate components: eriodictyol, and apigenin-7-O-glucoside were reported to induce the nodulation genes of R. leguminosarum. In addition, molecules having structures related to those of the inducer found in exudate were assessed for their ability to induce. Inducers of Rhizobium nodulation genes appear in general to be limited to certain substituted flavonoids, and the range of compounds to which a Rhizobium responds is species specific. Since host range is used to classify Rhizobium strains into different species, this suggests that differential response to inducer molecules is involved in the mechanism of determination of host range.
Two isoflavone components of soybean exudate, daidzein and genistein, have been reported to be inducers of the nodulation genes of B. japonicum strains 110 and 123 (KosslaK et al. (1987) Proc. Natl. Acad. Sci. USA 84:7423-7432). Several other isoflavones were found to be inducers (7-hydroxyisoflavone, 5,7-dihydroxyisoflavone and biochanin A) or weak inducers (formononetin and prunetin) of the B. japonicum nod genes. In addition, two flavones: 4',7-dihydroxyflavone and apigenin which induce certain Rhizobium nod genes were also found to induce the B. japonicum nod genes.
In view of the above, it is clear that the exchange of signals between legume and bacterial strain and intricacies thereof are shared between different legumes and the Rhizobium and Bradyrhizobium generas.
The manner in which nodulation genes are regulated is also conserved among Rhizobium and Bradyrhizobium strains.
Soybean Glycine max (L.) Merr.! is the world's most widely-produced nitrogen (N) fixing crop. However, soybean is a plant of tropical to subtropical origin and, as such, requires temperatures in the 25 to 30.degree. C. range for optimal growth and symbiotic N.sub.2 fixation. When well-nodulated, soybean is capable of fixing its own N. Both symbiotic N.sub.2 fixation and NO.sub.3 -utilization appear to be essential for maximum yield. High soybean yields also require adequate levels of phosphorous and potassium. Liming acid soils to a pH of 6.0 to 6.5 is an important prerequisite for profitable soybean production. Adequate populations of Bradyrhizobium japonicum must be present to produce a well-nodulated soybean crop. Smith et al. (1981) determined that an inoculum level above 1.times.10.sup.5 rhizobia per centimetre of row was necessary to establish effective nodulation.
Root zone temperatures (RZTs) below 25.degree. C. strongly and negatively affect soybean nodulation and N.sub.2 fixation (Lynch and Smith, 1994). In fact, in short season areas low temperature is considered the major growth limiting factor for soybean. It has been noted that all stages of nodule formation and functioning are affected by suboptimal RZTs and experiments have generally indicated that early nodule development processes are the most sensitive. The exact mechanism by which suboptimal RZTs affect N fixation has yet to be identified. Numerous hypothesis have been postulated however: 1) decrease N fixation activity by the nitrogenase enzyme complex; 2) changes in nodule oxygen permeability; 3) rate of export of fixed N from the nodule; 4) inhibition of N.sub.2 fixation inside the nodule; 5) decrease in bacteroid tissue and/or delay in its rate of formation; 6) via effects on bacterial physiology and growth; and 7) via effects on plant physiology and growth.
Production or N fertilizer, in Canada as elsewhere, is economically ($1 billion per year in Canada), energetically (equivalent to 30 million barrels of oil per year) and environmentally (produce 15 million tones of CO.sub.2 per year, ground water-polluting NO.sub.3 and ozone-destroying NO.sub.x) expensive. In eastern Canada the farm community spends approximately $150.times.10.sup.6 per year for N fertilizer. Nitrogen fixation is the sustainable alternative to N fertilizer. Therefore, an understanding of the mechanism of suboptimal RZT effects on soybean nodulation and N.sub.2 fixation and finding methods to reduce this restriction by low RZT would allow increased use of this N.sub.2 -fixing cash crop, and decreased reliance on potentially polluting N fertilizers in cool season areas. The ability to overcome the negative effects of suboptimal RZTs could be applied to other conditions that negatively affect nitrogen fixation (water stress, high pH, temperatures etc.).
Due to the number of benefits which can result from the establishment of rhizobia:legume symbiosis, a number of strategies have been devised to promote nodulation of legumes.
U.S. Pat. No. 4,878,936 to Handelsman et al., teaches a method for enhancing nodulation of legumes which includes inoculation in the immediate vicinity of the roots thereof, an effective quantity of bacteria which enhance nodulation. However, the results are based on controlled laboratory conditions, not on field studies. Moreover, the laboratory conditions used, involved temperatures above 25.degree. C. which are not expected to be limiting for nodulation.
U.S. Pat. No. 5,141,745 to Rolfe et al., discloses flavones, some of which are leguminous plant exudates, which induce expression of certain nod genes in rhizobium strains. Rolfe et al., however, do not assess whether their results, all obtained under laboratory conditions, translate into increase nodulation and growth of the leguminous plant under field conditions.
The art is replete with examples demonstrating that results obtained under the laboratory setting are not predictive of the field situation. Typically, a good controlled environment provides optimal levels of soil nutrients, soil pH, soil moisture, air humidity, temperature and light. The plants are usually widely spaced so that they do not compete for light and the light intensity is usually high. In some cases environmental factors such as carbon dioxide may even be optimized. The field environment is vastly more complicated than that of the controlled environment setting. The soil will vary in its chemistry and texture in a fractal pattern, such that, while the soil of a research site can be characterized in general, it will be variable at every level within the confines of the experimental area. In a controlled environment setting plants are usually produced in sterilized rooting media (pasteurized soil, sterile sand, or some form of artificial rooting media) and there is no soil micro flora or fauna. Field soil is an ecosystem; it contains an enormous number of bacteria, fungi, protista, algae, and soil insects. The climate and related atmospheric factors (light intensity, relative humidity, temperature, rainfall, carbon dioxide concentration of the air, presence of pollutants) vary constantly under unpredictably field conditions. Thus, a researcher may impose a nutrient limitation in the field, but if the conditions are dry and water is more limiting to plant growth than the nutrient in question, there will be no discernable effect due to nutrient treatments.
The inability to extrapolate from a laboratory to a field setting is illustrated by work conducted in the 1970's and early 1980's on soybean with strains of Bradyrhizobium japonicum which were hypothesized to be more energy efficient when fixing nitrogen. Because of the extreme stability of the triple bond in the dinitrogen molecule nitrogen fixation was known to be a very energy expensive process (reviewed In Schubert 1982). In addition, it was discovered that the enzyme which fixed dinitrogen into biologically useful ammonia (nitrogenase) leaked high energy electrons to protons, so that every time one dinitrogen molecule was fixed into two ammonia molecules, at least one dihydrogen (the product of two protons plus two electrons) was produced. This constituted a waste of energy by the plant-bacterium symbiotic system. Shortly afterward it was discovered that some strains of B. japonicum contained an enzyme that took up the hydrogen formed and took the high energy electrons back off the protons, hence recovering much of the energy that would have been lost (Schubert et al, 1978). This lead to speculation that strains containing these "uptake hydrogenases", referred to as Hup+ strains, would be more efficient and lead to improved plant growth, as the plant would have to supply less energy (as organic acids) to the bacteria for each ammonia molecule received from them. Albrecht et al. (1979) compared soybean plants inoculated with Hup+ and Hup- strains of B. japonicum under greenhouse conditions. Average total nitrogen contents and total dry weights of Hup+ inoculated plants were shown to be larger than those of plants inoculated with Hup- strains. This was confirmed by Maier et al. (1978). However, under field conditions, Albrecht et al. (1979) were unable to detect an increase in dry matter production or yield between Hup+ and Hup- strains. These results were confirmed by numerous field condition studies. During the course of these confirmations however, a superior strain of B. japonicum (532c), which is now included in almost all soybean inoculants used to produce soybean in Canada, was identified (Hume et al., 1990). Strikingly, this strain is Hup-.
This example provides a blatant proof involving soybean, that results obtained in a controlled milieu are a priori not predictive of the field situation.
U.S. Pat. No. 5,175,149 of Stacey et al., teaches that the mere coating of the leguminous seeds or sowing of the soil with the desired bacterial strins does not necessarily lead to the desired inoculation of the plant. Therefore, they provide a means for inducing nodulation on the roots of leguminous plants that is independent of the presence of rhizobial bacteria, by using the bacterial signal molecule directly (lipo-oligosaccharide), thereby by-passing the plant signal molecule (flavonoids).
U.S. Pat. No. 5,229,113 ('113) issued to Kosslak et al., relates to nodulation-inducing compositions and methods of selectively activating nod genes under the control of a soybean exudate inducible promoter responsive to inducer molecules. Similarly to U.S. Pat. No. 5,141,745, '113 does not teach or suggest that their compositions and methods are operational under field conditions and/or under conditions that inhibit or delay nodulation.
PCT patent application WO 94/25568, which was published Nov. 10, 1994 in the name of Rice et al., discloses cold tolerant strains of Rhizobium which are useful for improving nodulation, nitrogen fixation and overall crop size under field conditions. However, it is unclear whether the cold-selected strains indeed provided an advantage to Alfalfa, since in certain experiments the temperate strains performed better than the cold-temperature selected strain (i.e. Tables 5,6 and 7). This results corroborates the findings of Lynch et al., 1994 which suggested that inoculation with B. Japonicum strains from cold environments is unlikely to enhance soybean N2-fixation under cool soil conditions. Lynch et al., 1994 also suggested that indeed the host plant, and not the bacterial strain, mediates at least a significant portion of the sensitivity of N2-fixation under low RZT. Further WO 94/25568 (see below) teaches that commercial rhizobial inoculants are not consistent in their efficacity and performance, and nodulation failures after use of commercial inoculants are common. This is explained by the inability of inoculant strains to out-compete indegenous rhizobial bacteria for root-infection sites, once again demonstrating the non-predictivity of lab results to the field conditions. In any event WO 94/25568 fails to provide any teaching or suggestion as to involvement of the signal molecules in the initiation of the nodulation event and their effect under field conditions.
U.S. Pat. No. 5,432,079 to Johansen et al., relates to the isolation of Rhizobium strains having improved symbiotic properties. Once again this Patent falls to teach an enahancement of growth and or yield of a legume under field conditions. Moreover, this document is silent on the use of flavonoids or the like to achieve that goal. It teaches however that a higher expression of the nod genes does not necessarily provide an advantage, but can be detrimental to the competitive ability of the Rhizobium strains.
To date there has been no investigation as to whether nodulation inbiting or delaying factors, such as suboptimal RZTs alter plant to bacteria signaling.
An understanding of the mechanism of suboptimal RZT effects on soybean nodulation and N.sub.2 fixation and finding methods to reduce this restriction by low RZT would allow increased use of this N.sub.2 -fixing cash crop, and decreased reliance on potentially polluting N fertilizers in cool season areas. Elucidation of the mechanism for suboptimal RZTs effects on nodulation and nodule formation in soybean and a determination of how to reduce the negative effect of suboptimal RZTs on the soybean. N.sub.2 fixation symbiosis under the cool spring conditions or other conditions which inhibit or delay this symbiosis would provide a significant advantage to the production of legumes. For example, it would be advantageous to understand whether the poor nodulation of soybean at suboptimal RZTs are related to the plant's ability to synthesize and/or excrete plant-to-bacterial signal molecules.
There thus remains a need to reduce the the negative effects of environmental factors on nodulation and nodule formation and to provide compositions and methods to enable the enhancement of grain yield and protein yield of legumes grown under environmental conditions that inhibit or delay nodulation thereof.
The description found hereinbelow refers to a number of documents, the content of which is herein incorporated by reference.
Recent reviews on nodulation factors and Rhizobium symbiosis are available: Spaink, 1995, "Molecular basis of injection and nodulation by Rhyzobia--the ins- and outs of sympathogenesis". Ann. Rev. Phytopathol. 33:345-368; and Prome et al., 1996, "Nodulation factors in plant microbe interactions, Ed. Stacey et al., Pub. Chapman and Hall.